Immunogenic LHRH compositions and methods relating thereto

ABSTRACT

The present invention relates generally to an immunogenic LHRH composition and more particularly to an immunogenic LHRH composition comprising a LHRH C-terminal fragment of at least five amino acids. The present invention is useful, inter alia, as a prophylactic and/or therapeutic agent for the modification of fertility and behaviour patterns of animals, the achievement of livestock production gains such as increasing growth, decreasing feed conversion ratios or the control of unwanted organoleptic characteristics or the treatment of disorders of the reproductive organs.

The present invention relates generally to an immunogenic LHRHcomposition and more particularly to an immunogenic LHRH compositioncomprising a LHRH C-terminal fragment of at least five amino acids. Thepresent invention is useful, inter alia, as a prophylactic and/ortherapeutic agent for the modification of fertility and behaviourpatterns of animals, the achievement of livestock production gains suchas increasing growth, decreasing feed conversion ratios or the controlof unwanted organoleptic characteristics or the treatment of disordersof the reproductive organs.

Bibliographic details of the publications referred to by author in thisspecification are collated at the end of the description. SequenceIdentity Numbers (SEQ ID NOs.) for the amino acid sequences referred toin the specification are defined following the bibliography.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

Vaccination against the hypothalamic hormone luteinising hormonereleasing hormone (referred to herein as “LHRH”, also known as GnRH) hasbeen demonstrated as an immunological method of controlling reproductionsince the early 1970's (Fraser 1975, Jeffcoate et al 1982). Eliciting animmune response to LHRH prevents the release from the anterior pituitaryof the hormones LH and FSH, which are required for the development andmaintenance of the gonads—the testes in the male and ovaries in thefemale. Thus reduction of LH and FSH levels leads to loss ofreproductive function.

De-sexing (or neutering) operations are the most widely practisedsurgical procedures in veterinary medicine and livestock animalmanagement. A significant proportion of both sexes of domestic livestockand companion animals are routinely surgically de-sexed to prevent avariety of undesirable characteristics which accompany sexual maturity.The traits include fighting, wandering, sexual behaviour, loss ofcondition, tumours of reproductive organs and pregnancy.

The control of mating behaviour by vaccination with LHRH-conjugatevaccines in companion animals such as dogs, cats and horses, and inlivestock specifically in male pigs and male and female cattle, has beenidentified by the inventor as a goal as significant as the control offertility.

Similarly, the control and treatment of disorders of the gonads andother reproductive organs, of both humans and animals, such astesticular cancer, breast cancer, prostate cancer, ovarian cancer,prostate enlargement or endometriosis is of significance.

The first published report of vaccination with an LHRH conjugate vaccinein rabbits showed that a dramatic effect was achieved in the developmentof the testes. Early reports of the application of an LHRH vaccine inpigs (Falvo et al, 1986, Caraty and Bonneau 1986), showed that effectiveformulations based on 1-10 LHRH conjugated to human serum globulin orbovine serum albumin could control testes development and boar taint.Awonyi et al. (1988) showed that the effect of vaccination of pigsagainst LHRH affected primarily testis development. All these trialswere done on small numbers of animals, with no reports of efficacy.

The problems of variability of LHRH-conjugate vaccines in controllingboar taint have been attempted to be overcome by geneticallyincorporating LHRH amino acid sequences into carrier proteins, includingthe pilin gene from E. coli (Zee et al 1995) and into a truncatedleucotoxin gene from Pasteurella haemolytica (Potter et al 1997). Thesefusion proteins are produced as recombinant molecules and not bybiochemical coupling. Trials have shown these recombinant proteins tofunction as immunocastration vaccines. However, they have not resultedin commercially available vaccines and press reports suggest less thandesired efficacy.

In keeping with the less than perfect nature of highly developed andwidely applied subunit vaccines for disease prevention, immunocastrationvaccines based on specific LHRH-protein conjugates have also been shownto be less than perfect at inducing antibody to LHRH or in reducinghormones or other parameters associated with reproductive functions.There has been a general recognition of a wide variation in theeffective induction of antibody to LHRH with a variety of LHRH-conjugatevaccines (Meloen et al 1994).

Vaccination of cattle with a 1-10 LHRH peptide-human serum albuminconjugate in Freunds adjuvant (Robertson et al, 1982), gave goodantibody responses to LHRH after 2 vaccinations in only 5 of 10vaccinated cattle. Even with boost vaccinations, the poor responders didnot maintain antibody titres or have suppressed testosterone. Acommercially developed vaccine for cattle (Vaxstrate), was only 80%effective (Hoskinson et al 1990).

Experiments in mice (Sad et al 1991) have shown that responses toLHRH-conjugates are genetically based. The vaccine was a 1-10 LHRHpeptide, with the substitution of D-lysine instead of L-glycine at the 6position, conjugated to diphtheria toxoid and adjuvanted with alum. Somestrains of mice responded well, while others showed suppression ofantibody to LHRH. These results would lead those skilled in the art ofvaccine formulation to expect that a significant proportion of anoutbred population would fail to respond or respond poorly to anLHRH-conjugate subunit type vaccine.

Vaccination of male pigs has resulted in variable suppression of testisdevelopment and suppression of boar taint. Bonneau and coworkers haveshown (Bonneau et al 1994) that a 1-10 LHRH-α globulin conjugate givenin oil emulsion for primary vaccination and saponin adjuvant for boostvaccination gave an antibody response in only 90% of 20 vaccinated pigs.Testosterone levels were suppressed in only 16/20 vaccinates (75%). Thusthe quality as well as the amount of antibody is important indetermining the efficacy of an LHRH-conjugate based vaccine. Hagen et al(1988) claimed that vaccination of 6 boars with an LHRH-bovine serumalbumin (BSA) conjugate in Freunds adjuvant could reduce boar taint.However, 2/6 boars had low antibody responses and had normalspermatogenesis and testis function. Skatole levels were not affected byvaccination against LHRH. Accordingly, there is a need to develop anLHRH vaccine which is consistently more highly effective than thoseutilized to date.

In work leading up to the present invention, the inventor has determinedthat the efficacy of vaccination against LHRH is significantly improvedwhen LHRH is administered as a conjugate with diphtheria toxoid and anionic polysaccharide.

Accordingly, one aspect of the present invention relates to apreparation for use in eliciting an effective immune response to LHRH,said preparation comprising a LHRH-diphtheria toxoid conjugate adsorbedto an ionic polysaccharide.

Reference to an “ionic polysaccharide” should be understood as areference to any positively or negatively charged polysaccharide orderivative or chemical equivalent thereof. Reference to “derivative” and“chemical equivalent” should be understood to have the same meaning asoutlined below. Said ionic polysaccharide may be in soluble or insolubleform. Preferably said ionic polysaccharide is an ionic dextran. Evenmore preferably said ionic dextran is DEAE-dextran, dextran sulphate orQAE-dextran. Most preferably, said ionic dextran is DEAE dextan.Preferably, the dextran component of said ionic dextran exhibits amolecular weight in the range 250,000 to 4,000,000 Da and even morepreferably 500,000 to 1,500,000 Da.

According to this most preferred embodiment, the present inventionrelates to a preparation for use in eliciting an effective immuneresponse to LHRH, said preparation comprising a LHRH-diphtheria toxoidconjugate adsorbed to DEAE-dextran.

Reference to an “effective” immune response should be understood as areference to an immune response which either directly or indirectlyleads to the reduction or complete blocking of reproductive function(i.e. reduces or prevents the functioning of any one or more of thereproductive organ's activities or modulates the hormonal levels of ananimal such that any one or more aspects of reproduction or reproductiveactivity are reduced) in at least 90%, and preferably at least 95%, ofanimals treated. It should be understood that efficacy is a functionalmeasure and is not defined by reference to anti-LHRH antibody titrealone since the presence of circulating antibody alone is notnecessarily indicative of the capacity of said circulating antibody toblock reproductive function. The term “reproductive organ” should beunderstood in its broadest sense to refer to the male and female gonadsand accessory sexual organs. “Accessory sexual organs” should also beunderstood in its broadest sense and includes, for example, theprostate, breasts and the uterus.

Reference hereinafter to “LHRH” should be read as including reference toall forms of LHRH and derivatives thereof.

“Derivatives” include fragments, parts, portions, chemical equivalents,mutants, homologs and analogs from natural, synthetic or recombinantsources, including fusion proteins. For example, said LHRH includespeptides comprising a sequence of amino acids substantially as set forthin SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or havingat least 50% similarity thereto. The molecules defined in SEQ ID Nos:1,2 and 3 are from the human and are conserved across all mammals. SEQ IDNO:4 is a derivative of SEQ ID NO:2 wherein spacers have been introducedat the N-terminus. Chemical equivalents of LHRH can act as a functionalanalog of LHRH. Chemical equivalents may not necessarily be derived fromLHRH but may share certain similarities. Alternatively, chemicalequivalents may be specifically designed to mimic certain physiochemicalproperties of LHRH. Chemical equivalents may be chemically synthesisedor may be detected following, for example, natural product screening.

Homologs of LHRH contemplated herein include, but are not limited to,LHRH derived from different phyla including birds, fish, reptiles andinvertebrates.

“Derivatives” may also be derived from insertion, deletion orsubstitution of amino acids. Amino acid insertional derivatives includeamino and/or carboxylic terminal fusions as well as intrasequenceinsertions of single or multiple amino acids. Insertional amino acidssequence variants are those in which one or more amino acid ornon-natural amino acid residues are introduced into a predetermined sitein the protein although random insertion is also possible with suitablescreening of the resulting product. Deletional variants arecharacterised by the removal of one or more amino acids from sequence.Substitutional amino acid variants are those in which at least oneresidue in the sequence has been removed and a different natural ornon-natural residue inserted in its place. Typical substitutions arethose made in accordance with Table 1:

TABLE 1 Suitable residues for ammo acid substitutions Original ResidueExemplary Substitutions Ala Ser * Arg Lys Asn Gln; His Asp Glu Cys SerGln Asn * Glu Ala * Gly Pro * His Asn; Gln Ile Leu; Val * Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr * Ser Thr Thr Ser * TrpTyr * Tyr Trp; Phe Val Ile; Leu Key: Amino acid residues marked with anasterisk indicate residues present in the mammalian LHRH sequence.

Examples of non-natural amino acids include, but are not limited to theD-isomers of said amino acids. “Additions” to amino acid sequencesinclude fusion with other peptides, polypeptides or proteins.

Reference to diphtheria toxoid should be understood as a reference toall forms of diphtheria toxoid and derivatives thereof. The term“derivatives” has the same meaning as hereinbefore defined. Derivativesmay include, for example, molecules comprising the diphtheria toxoid Tcell epitopes or said T cell epitopes in isolation.

Preferably, said LHRH comprises an LHRH C-terminal fragment of at leastfive amino acids. Even more preferably, said LHRH comprises the aminoacid sequence substantially as set forth in SEQ ID NO:2 and wherein thecarboxyl terminus of said amino acid sequence is amidated. Saidpreferred LHRH is referred to herein as “LHRH 2-10 form”.

According to this most preferred embodiment there is provided apreparation for use in eliciting an effective immune response to LHRHsaid preparation comprising a LHRH 2-10 form-diphtheria toxoid conjugateadsorbed to DEAE dextran.

In another preferred embodiment said LHRH comprises the amino acidsequence substantially as set forth in SEQ ID NO:4. Said preferred LHRHis referred to herein as “modified LHRH 2-10 form”.

According to this preferred embodiment there is provided a preparationfor use in eliciting an effective immune response to LHRH saidpreparation comprising a modified LHRH 2-10 diphtheria toxoid conjugateadsorbed to DEAE dextran.

Although not intending to limit the invention to any one method, saidpeptide may be synthesised by Fmoc chemistry and the resulting peptidecoupled to the carrier protein diphtheria toxoid. Said coupling may beperformed as described in Ladd et al 1990 or in Bonneau et al 1994, andthe resulting conjugate of peptide and carrier protein (referred toherein as “peptide-conjugate”) processed to be free of unbound peptideand other biproducts of conjugation. Such processing may be achieved byconventional dialysis or by ultrafiltration. The resultingpeptide-conjugate is adsorbed to the ionic polysaccharide adjuvant.

Without limiting the present invention to any one theory or mode ofaction, administration of an effective amount of the LHRH preparation ofthe present invention induces a significantly more effective immuneresponse against LHRH than the LHRH-carrier-adjuvant compositionsdescribed in the prior art. Said improved efficacy is observed when theimmunogenic LHRH composition specifically comprises the carrierdiphtheria-toxoid and an ionic polysaccharide adjuvant.

In another aspect of the present invention there is provided apharmaceutical composition comprising a LHRH-diphtheria toxoid conjugateadsorbed to an ionic polysaccharide together with one or morepharmaceutically acceptable carriers and/or diluents.

Preferably said ionic polysaccharide is DEAE dextran.

Even more preferably said LHRH is the LHRH 2-10 form.

As used herein, the term “pharmaceutical” includes “veterinary”.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption or delayed release ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying, the freeze-dryingtechnique and the spray-drying technique which yield a powder of theactive ingredients plus any additional desired ingredient frompreviously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or they may be enclosed in hard or soft shell gelatincapsule, or compressed into tablets, or incorporated directly with thefood of the diet. For oral administration, the active compound may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least1% by weight of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 5 to about 80% of the weight of the unit. The amount of activecompound in such useful compositions is such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 0.1 μg and 2000 μg of active compound.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter: A binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; and a lubricant such as magnesium stearate. When thedosage unit form is a capsule, it may contain, in addition to materialsof the above type, a liquid carrier. Various other materials may bepresent as coatings or to otherwise modify the physical form of thedosage unit. A syrup or elixir may contain the active compound, methyland propylparabens as preservatives, and a dye. Of course, any materialused in preparing any dosage unit form should be veterinarily pure andsubstantially non-toxic in the amounts employed. In addition, the activecompound(s) may be incorporated into sustained-release preparations andformulations.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for veterinarily active substances is well knownin the art Except insofar as any conventional media or agent isincompatible with the active ingredient; use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.For administration to livestock it is particularly advantageous to use amulti-dose container linked to a repeating vaccination gun. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subjects to be treated; each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the novel dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from 0.5 μg to about 2000 μg.Expressed in proportions, the active compound is generally present infrom about 0.5 μg to about 2000 μg/ml of carrier. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

Although not intending to limit the invention to any one theory or modeof action, the induction of an effective immune response against LHRHresults in prevention of the release of the hormones LH and FSH from theanterior pituitary. Since these hormones are required for thedevelopment and maintenance of the gonads, reduction in the levels ofthese hormones leads to a decrease or loss of reproductive functions.The vaccinated animals are therefore effectively neutered resulting inthe loss of characteristics associated with sexual maturity such asfighting, wandering, sexual behaviour, loss of condition, organolepticcharacteristics, tumours of reproductive organs and pregnancy.

Accordingly, another aspect of the present invention relates to a methodof eliciting, in an animal, an effective immune response to LHRH saidmethod comprising administering to said animal an effective amount ofLHRH-conjugate.

Reference to “LHRH-conjugate” should be understood as a reference to theLHRH preparation of the present invention.

Reference to “animal” should be understood as the reference to allanimals including primates (e.g. humans, monkeys), livestock animals(e.g. sheep, cows, horses, donkeys, goats, pigs), laboratory testanimals (e.g. rats, guinea pigs, rabbits, hamsters), companion animals(e.g. dogs, cats), captive wild animals (e.g. emus, kangaroos, deer,foxes), aves (e.g. chickens, ducks, bantams, pheasants, emus,ostriches), reptiles (e.g. lizards, snakes, frogs) and fish (e.g. trout,salmon). Said animal may be male or female.

In a most preferred embodiment, the present invention relates to amethod of eliciting, in an animal, an effective immune response to LHRHsaid method comprising administering to said animal an effective amountof LHRH-conjugate wherein said immune response inhibits the reproductivecapacity of said animal.

Preferably said LHRH-conjugate is LHRH 2-10 form.

Reference to “inhibiting the reproductive capacity of an animal” shouldbe understood as the partial or complete reduction of the functioning ofany one or more of the reproductive organs's activities or modulation ofsaid animal's hormonal levels such that reproductive activity, such assexual activity, is reduced.

Inhibiting the reproductive capacity of an animal may result in a numberof consequences such as, but not limited to, the castration of saidanimal or the reduction or elimination of characteristics associatedwith sexual maturity (for example, fighting, wandering, sexualbehaviour, loss of condition, unwanted organoleptic characteristics,tumours of reproductive organs and pregnancy). “Castration” should beunderstood as a reference to the neutering of both male and femaleanimals. Inhibiting the reproductive capacity of an animal may alsoresult in the cessation of tumor cell proliferation (for e.g. prostatecancer cells, breast cancer cells, ovarian cancer cells or testicularcancer cells), inhibition or reversal of hyperplasia, such as prostateenlargement, endometriosis or inflammatory responses.

Accordingly, another aspect of the present invention relates to a methodof castrating an animal said method comprising administering to saidanimal an effective amount of LHRH-conjugate.

Preferably said LHRH-conjugate is the LHRH 2-10 form.

Yet another aspect of the present invention relates to a method ofregulating oestrus cycling in a female animal said method comprisingadministering to said animal an effective amount of LHRH-conjugate.

Preferably said LHRH-conjugate is the LHRH 2-10 form.

Reference to “regulating” should be understood in its broadest sense andincludes, for example, inhibiting or delaying oestrus.

Still yet another aspect of the present invention relates to a method ofinhibiting characteristics induced by the sexual maturation of an animalsaid method comprising administering to said animal an effective amountof LHRH-conjugate.

Preferably said LHRH-conjugate is the LHRH 2-10 form.

Reference to “inhibiting characteristics induced by the sexualmaturation of an animal” should be understood as a reference to thereduction or complete elimination of any one or more physical and/orbehavioural characteristics induced either directly or indirectly bysexual maturation. Said physical and/or behavioural characteristicsinclude, for example, fighting, wandering, sexual behaviour, loss ofcondition, unwanted organoleptic characteristics, oestrus cycling,fertility, pregnancy and tumours of the reproductive organs.Accordingly, inhibiting said characteristics includes inhibiting sexualactivity (for example preventing female cattle mounting other femalecattle) preventing or delaying ovulation, reducing aggressive behaviouror reducing unwanted organoleptic characteristics such as boar taint. Ina particularly preferred embodiment, said characteristics are aggressionand sexual activity.

According to this preferred embodiment there is provided a method ofinhibiting aggression in an animal said method comprising administeringto said animal an effective amount of LHRH 2-10 form-conjugate.

In another most preferred embodiment there is provided a method ofinhibiting sexual activity in an animal said method comprisingadministering to said animal an effective amount of LHRH 2-10form-conjugate.

Vaccination with LHRH conjugate in male dogs and cats can be used tocontrol unwanted behaviour, particularly aggression and the urge toroam. In female dogs and cats, the desired effects are control offertility and of unwanted behaviour at the time of oestrus, commonlytermed “in heat” or “in season”. The unwanted behaviour in femalesincludes increased fractiousness, marking of territories, wandering andother behaviours associated with oestrus.

According to this most preferred embodiment there is provided a methodof inhibiting characteristics induced by the sexual maturation of catsand/or dogs said method comprising administering to said cat and/or dogan effective amount of LHRH-conjugate.

Most preferably said characteristics are aggression and roaming in malecats and/or dogs and fractiousness, marking of territory, wandering andoestrus behaviour in female cats and/or dogs.

In the thoroughbred horse industry, the racing of stallions isassociated with difficulty in handling and ease and consistency oftraining. A large proportion of young colts are gelded and raised ascastrates to make them more manageable. This does not appear to impactsignificantly on their racing potential. A vaccine to control unwantedbehavioural problems would allow the full racing potential of malehorses to be realised, with the added benefit of reversibility and soobtaining the genetic benefit as a stud animal after their racing careeris over.

The racing of fillies and mares (female horses) is at its height in thespring and to some degree in the autumn in the temperate climates of theworld. It is at these times of the year that horses come into season.This causes difficulties in training, handling and in uneven and poorracing performance. An LHRH vaccine to control oestrus would have alarge and ready market in the horse racing industry. There are currentlyproducts based on hormone analogues available to control oestrus inracing fillies and mares. These are reported to have a lasting effect onthe ability of treated mares to breed.

Accordingly, in yet another preferred embodiment there is provided amethod of inhibiting characteristics induced by the sexual maturation ofhorses said method comprising administering to said horse an effectiveamount of LHRH-conjugate.

Most preferably said characteristics are aggression in colts and oestrusbehaviour and uneven performance in mares.

In cattle, the unmanageable behaviour of bulls is well known. Aggressionof bulls can be directed toward stockmen, inanimate objects such asfences and drinking troughs and can result in serious fighting betweencattle. Thus in most beef producing countries, bulls destined for beefproduction are castrated while still calves, and the resulting steersare raised. The raising of steers in preference to entire males has asignificant negative impact on production performance, but this isjudged to be an acceptable, even necessary trade off over the raising ofmore docile steers.

Heifers are raised for beef production in the USA and in Australia. Thecycling of heifers in feedlots causes significant production losses. Thecycling heifer has a large increase in activity levels, resulting inpoor or negative growth over the 5-7 days of the cycle. The heightenedactivity levels of heifers in oestrus impacts on other heifers in thesame pen, so that the production performance of the entire pen of 50100animals is affected. In the USA heifers are fed a diet containingmelengestrol acetate (MGA), a synthetic steroid, to control oestrus. InAustralia, and other countries where hormonal feed supplements areprohibited, heifers are raised in feedlots without feeding of MGA, withpoor production performance.

Accordingly, the immunocastration of livestock, although reducing oreliminating characteristics associated with sexual maturity, generallyresults in a negative impact on production gains on immunocastratedanimals over uncastrated animals. This theory is based on the wellestablished fact that entire animals grow considerably faster and moreefficiently than castrate animals. However, the inventor has determinedthat administering the LHRH preparation of the present invention tolivestock nevertheless results in the achievement of production gains.Reference herein to “production gains” includes but is not limited to anincrease in final weight of livestock at slaughter, lowering of feedrequirements for each kilogram of carcass weight gained, increasinggrowth rate of said livestock as compared to uncastrated livestock,improving the quality of meat derived from said livestock (for example,by controlling unwanted organoleptic characteristics of said meat) ordecreasing stress levels of intensively housed livestock by reducingaggressive interactions of the intensively housed animals or, withrespect to pigs, control of boar taint.

Accordingly, yet another aspect of the present invention relates to amethod of achieving production gains in livestock said method comprisingadministering to said livestock an effective amount of LHRH-conjugate.

Preferably said production gain is the reduction or elimination ofunwanted organoleptic characteristics of meat from male livestock.

The LHRH-conjugate may be administered to the livestock in asingle-dose, for example a single administration of a slow or pulsatilerelease vaccine or in multiple doses.

Preferably said LHRH-conjugate is the LHRH 2-10 form.

Accordingly, there is provided a method of achieving production gains inlivestock said method comprising administering to said livestock aneffective amount of an LHRH 2-10 form-conjugate.

Preferably said production gain is the reduction or elimination ofunwanted organoleptic characteristics of meat from male livestock.

The term “livestock” includes but is not limited to mammals such aspigs, cattle, sheep; captive wild animals such as deer; and aves such asemus or ostriches. Most preferably, said livestock are pigs and cattle.

According to this most preferred embodiment, there is provided a methodof achieving production gains in pigs said method comprisingadministering to said pigs an effective amount of an LHRH 2-10form-conjugate.

Preferably said production gain is the reduction or elimination of boartaint.

In another most preferred embodiment, there is provided a method ofachieving production gains in cattle said method comprisingadministering to said cattle an effective amount of an LHRH 2-10form-conjugate.

In animals, and particularly humans, vaccination with LHRH-conjugate canbe used as a prophylactic or therapeutic treatment for disorders whichare modulated directly or in directly by LHRH. These disorders includemalignancies of cells which are regulated by LHRH or regulated byhormones which are themselves regulated by LHRH, for example, testicularcancer, breast cancer, ovarian cancer, prostate cancer and cancers ofoncofoetal cells or cells which are induced to express oncofoetalantigens when malignancy occurs. These disorders also include nonmalignant proliferative disorders such as hyperplasias, for example,prostatic hyperplasia or endometrial hyperplasia. Without limiting thepresent invention to any one theory or mode of action, some tumor cellsexpress receptors for reproductive hormones, the synthesis of which areregulated by LHRH. By vaccinating against LHRH it is possible to preventthe release of these hormones. The LHRH-conjugate of the presentinvention may also be used to treat or prevent disorders such as ovarianpolycystitis, endometriosis and inflammatory conditions. Further uses ofthe LHRH-conjugate of the present invention include human fertilitytreatment based on modulation of the libido.

Accordingly, another aspect of the present invention relates to a methodof inhibiting the growth of cells which are regulated directly orindirectly by LHRH said method comprising administering an effectiveamount of LHRH-conjugate.

Preferably said cells are human cells.

Reference to cell “growth” is a reference to the proliferation,differentiation or functional activity of said cell. Reference to cellgrowth which is “regulated directly or indirectly by LHRH” should beunderstood as a reference to cell growth which is regulated by LHRHitself or cell growth which is regulated by hormones other than LHRHwhich are themselves either directly or indirectly regulated by LHRH.

Reference to “inhibiting” should be understood as a reference to theprevention of cell growth, the cessation of cell growth or the downregulation of cell growth. Said cells may be located within the organfrom which they derive or at some other location within the animal'sbody, such as, for example, where a malignant breast cell hasmetastasised in the liver.

In a particularly preferred embodiment said cells are malignant cellsand most particularly malignant testicular cells, malignant breastcells, malignant ovarian cells or malignant prostate cells.

In yet another preferred embodiment said cells are hyperplastic cellssuch as prostatic hyperplastic cells or endometrial hyperplastic cells.

In yet another aspect of the present invention there is provided amethod of down-regulating the libido of an animal said method comprisingadministering to said animal an effective amount of LHRH-conjugate.

Preferably said animal is a human.

Further features of the present invention are more fully described inthe following Examples. It is to be understood, however, that thisdetailed description is included solely for the purposes of exemplifyingthe present invention. It should not be understood in any way as arestriction on the broad description of the invention as set out above.

EXAMPLE 1 Preparation of LHRH-Conjugate Composition

The LHRH-conjugate is based on a synthetic 2-10 form of LutenisingHormone Releasing Hormone (LHRH peptide coupled to a carrier protein.The peptide by itself is too small to be antigenic, and coupling to acarrier protein is required so that the peptide acts as a hapten andimmunity is induced to LHRH. The carrier protein is diphtheria-toxoid.

The peptide is synthesised by Fmoc chemistry and the resulting 2-10 formLHRH peptide is coupled to diphtheria toxoid. The coupling may beperformed as described in Ladd et al. 1990 or in Bonneau et al. 1994,and the resulting conjugate of peptide and diphtheria-toxoid processedto be free of unbound peptide and other by-products of conjugation. Suchprocessing may be achieved by conventional dialysis or byultrafiltration.

The resulting LHRH-carrier preparation may be used to prepare acomposition for administration by formulation with or in an adjuvant(referred to as “LHRH-conjugate”). The adjuvant is an ionicpolysaccharide such as DEAE-dextran, dextran sulphate or QAE-dextran.The adjuvant formulation may include a combination of two or more of theadjuvants listed. These lists are not to be taken as exhaustive. Theselection of adjuvant is in part dependant on the species being targetedand is based on the level and duration of the immune response requiredand on the lack of reactogenicity (ie tissue compatibility). The levelof active component and adjuvant are chosen to achieve the desired leveland duration of immune response.

Formulations of LHRH-conjugate suitable for use in the present inventionare preferably in the range of 5-500 μg of LHRH-diphtheria toxoid in5-500 mg of DEAE-dextran.

EXAMPLE 2

The LHRH vaccine (2-10 LHRH with diphtheria toxoid and DEAE dextranadjuvant as described in Example 1) was given to pigs as:

Group 1: 2 doses at 14 and 18 weeks of age, 10 male pigs per group.Group 2: 3 doses at 14, 18 and 22 weeks of age, 10 male pigs per group.Group 3: Controls received DEAE-dextran adjuvant alone, 10 male pigs pergroup.

Doses of LHRH vaccine were given subcutaneously. The LHRH vaccine ispreferably in the range of 50-500 μg of LHRH-diphtheria toxoid in 50-500mg of DEAE-dextran.

Pigs were slaughtered at 22 weeks of age (groups 1 and 3) or at 24 weeks(group 2).

Parameters Measured:

Anti-LHRH titres were measured at 2 weeks post 2nd dose.

Boat taint compounds skatole and androstenone were measured in fatsamples take at slaughter.

Results:

Anti-LHRH titres 2 weeks post boost. Group mean titres are shown.

Group Titre Group 1 4300 Group 2 2760 Group 3 <20 * Boar taint compoundswere measured in samples taken at slaughter.

Group mean values are shown.

Group Skatole* Androstenone* Group 1 0.06 0.18 Group 2 0.05 0.23 Group 30.07 0.51 *Boar taint compounds are expressed as μg/g fat tissue.

CONCLUSIONS

The LHRH vaccine induced high levels of antibody in all vaccinated pigsas determined at 2 weeks post boost.

The LHRH vaccine was able to control boar taint compounds in allvaccinated pigs.

EXAMPLE 3 Mice

10 mice were vaccinated with formulations consisting of analogues ofLEVI, linked to diphtheria toxoid and adjuvanted with DEAE dextran. Micewere vaccinated on days 0 and 14 and bled on day 21 to demonstrateinduction of antibody to LHRH.

Analogues of LHRH tested in mice include:

2-10 LHRH: His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂. (SEQ ID NO:2)Modified 2-10: Gly-Ser-Gly-Ser-Gly-Leu-Arg-Pro-Gly-NH₂. (SEQ ID NO:4)

Both constructs were linked to diphtheria toxoid by conventionalchemistries. Mice received between 5-50 μg of conjugate per injection in5-50 mg DEAE dextran adjuvant.

LHRH-conjugate titre 1 week post boost 2-10 - diphtheria toxoid 3005 (n= 9) Modified 2-10 1990 (n = 7)

Titres to LHRH were induced in all mice vaccinated with the aboveconstructs.

EXAMPLE 4 Cattle

Entire male and female cattle were vaccinated with a formulationcomprising 2-10 LHRH conjugated to diphtheria toxoid, and adjuvantedwith DEAE dextran. Cattle were 9-12 months of age at the time of initialvaccination Each dose contained between 50 and 500 μg conjugateformulated in DEAE-dextran (50-500 mg).

Vaccinations were at 0 days with a boost vaccination at 28 days. Bloodsamples were taken at monthly intervals after the boost vaccination, andantibody titres measured by ELISA.

Female cattle (heifers) behaviour was monitored by daily inspection bytrained farm staff and veterinarians. 8 weeks after boost vaccination,behaviour was also monitored by fixing of Heat Mount Detector pads(Kamar Marketing Group Inc, Steamboat Springs, Colo., USA, dye releasingpads) to the rump of heifers. Mounting or riding behaviour (also calledbulling) by cycling heifers will crush capsules of dye in the pads,which can be visualised from a distance. This usually only occurs whenthe standing heifer is receptive, ie in oestrus, and when the mountingheifer is also in oestrus. Thus the pads provide a useful continualmonitor of oestrus in vaccinated heifers run with control unvaccinatedheifers.

Results:

Anti-LHRH titres.

GMT=Geometric mean titre of group

GMT 3 weeks post GMT 7 weeks post Vaccination Group boost (range) boost(range) Placebo vaccinated controls <100 <100 LHRH-diphtheria toxoid10,357 3435 conjugate in DEAE dextran (3623-26133) (1538-15464) adjuvant

Riding Behaviour/Pad Reactivity:

Control heifers (female cattle) exhibited riding behaviour at the timeof primary and boost vaccination and during the first 7 weeks afterboost vaccination. None of the vaccinated cattle exhibited thebehavioural patterns associated with reproductive function in cyclingheifers. Scoring of cycling behaviour by direct observations wereconfirmed by Heat Mount Detector Pads, none of which were activated invaccinates during the 7 week post boost period.

These results confirm the ability of the preferred formulation vaccineto modify behaviour of vaccinated animals, in this example the controlof oestrus and associated behaviours in female cattle (heifers).

EXAMPLE 5 Dogs

Beagle/Foxhound cross dogs and bitches were vaccinated with aformulation comprising 2-10 LHRH conjugated to diphtheria toxoid, andadjuvanted with DEAE dextran. Dogs were 6-10 months of age at the timeof initial vaccination. Control dogs were not vaccinated.

Vaccinations were at 0 days with a boost vaccination at 28 days. Bloodsamples were taken at monthly intervals after the boost vaccination, andantibody titres measured by ELISA.

The dose of vaccine is preferably in the range of 50-500 μgLHRH-diphtheria toxoid in 10-100 mg DEAE-dextran.

Titres to LHRH in Dog Serum:

Weeks post boost Vaccinated with LHRH vaccination vaccine Unvaccinatedcontrols 0 <100 <100 4 84,640 <100 8 38,919 <100 12 7,900 <100

All titres shown are Geometric Mean Titres of the group of 7-8 dogs.Titres were measured by ELISA.

The data show that the favoured formulation of 2-10 LHRH conjugated todiphtheria toxoid in DEAE-dextran adjuvant induces a strong antibodyresponse in 100% of vaccinated dogs. Of significance in this example isthe demonstration that the preferred formulation is able to giveduration of the antibody response.

Inhibition of Development of Testes:

At 16 weeks post boost, testes sizes were measured in controls andvaccinates, by reference to orchidometer beads.

Testes Size (Group Mean Values):

Weeks post boost Vaccinated with LHRH vaccination vaccine Unvaccinatedcontrols 16 0.5 cm³ 12 cm³

These data demonstrate that the preferred formulation is able to preventthe development of reproductive organs, as shown in this example in theinhibition of the growth and maintenance of testes in dogs.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

-   Awonyi, C. A., Chandrashekar, V., Arthur, R. D., Schanbacher, B. D.    and Falvo, R. E. J. Androl. 9:160-171 (1988).-   Bonneau, M., Dufour, R, Chouvet, C., Roulet, C., Meadus, W. and    Squires, E. J. J. Animal Science 72: 14-20 (1994).-   Caraty, A. and Bonneau, M. C.R. Acad. Sci. Paris 303 Series    D:673-683 (1986).-   Falvo, R. E., Chandrashekar, V. et al. J. Animal Science 63: 986-994    (1986).-   Fraser, H. M. Immunization with Hormones in Reproductive Research:    07-116 (1975).-   Hagen, G., Andresen, O., Blichfeldt, T. and Berg, K. A. Proc. 11th    Congress on Animal Production Abstract 493 (1988).-   Hoskinson, R. M., Rigby, P. E., Mattner, V. L., Huynh, V. L.,    D'Occhio, M. D., Neish, A., Trigg, T. E., Moss, B. A., Lindsey, M.    J., Coleman, G. D. and Schwartzkoff, C. L. Aust. J. Biotechnol.    4:166-170 (1990),-   Jeffcoate, I. A., Lucas, J. M. and Crighton, D. B. Theriogenology    18:65-77 (1982).-   Ladd A., Tsong Y. Y., and Thau R. B., American J. Reproductive    Immunology 22: 56-63 (1990).-   Meloen, et al., Vaccine 12: 741-746 (1994).-   Potter, A. A. and Manns, J. G. Patent—International filing number    PCT/CA97/00559 (1997).-   Robertson, I. S., Fraser, H. M., Innes, G. M. and Jones, A. S. Vet.    Record 111:529-531 (1982).-   Sad S., Gupta H., Talwar G. P., and Raghupathy R, Immunology    74:223-227 (1991).-   Zee, A, Noordegraaf, C. V., Bosch, H., Gielen, J., Bergmans, H.,    Hoekstra, W. and Die, I. Vaccine 13:753-758 (1995).

1. A composition for use in eliciting an effective immune response toLHRH said composition comprising a LHRH-diphteria toxoid conjugateadsorbed to an ionic polysaccharide.
 2. The composition according toclaim 1 wherein said ionic polysaccharide is DEAE-dextran.
 3. Thecomposition according to claim 1 wherein said LHRH is LHRH 2-10 form. 4.The composition according to claim 1 wherein LHRH is modified LHRH 2-10form.
 5. A pharmaceutical composition comprising a LHRH-diphtheriatoxoid conjugate adsorbed to an ionic polysaccharide together with oneor more pharmaceutically acceptable carriers and/or diluents.
 6. Thepharmaceutical composition according to claim 5 wherein said ionicpolysaccharide is DEAE-dextran.
 7. The pharmaceutical compositionaccording to claim 5 wherein said LHRH is LHRH 2-10 form.
 8. Thepharmaceutical composition according to claim 5 wherein said LHRH ismodified LHRH 2-10 form.
 9. A method of eliciting, in an animal, aneffective immune response to LHRH said method comprising administeringto said animal an effective amount of the composition of claim 1.